Method for measuring binding kinetics with a resonating sensor

ABSTRACT

Detecting a presence of a subject material in a fluid sample using at least one resonating sensor immersible in the fluid sample. Binding kinetics of an interaction of an analyte material present in the fluid sample are measured with the resonating sensor, which has binding sites for the analyte material. Prior to exposing the resonating sensor to the fluid sample, operation of the resonating sensor is initiated, which produces a sensor output signal representing a resonance characteristic of the resonating sensor. Optionally, a reference resonator is used that produces a reference output signal. The reference resonator lacks binding sites for the analyte. Introduction of a fluid sample to the resonating sensor is automatically detected based on detection of a characteristic change in the sensor output signal or a reference output signal, or both. In response to the detecting of the introduction of the fluid sample, automated measurement of the binding kinetics of the analyte material to the resonating sensor are measured.

PRIOR APPLICATION

This application is a divisional application of U.S. patent applicationSer. No. 13/278,032, filed Oct. 20, 2011, now U.S. Pat. No. 8,409,875,which claims the benefit of U.S. Provisional Application No. 61/405,048filed Oct. 20, 2010. The disclosures of both applications areincorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to measurement and testing, and morespecifically to piezoelectric resonator sensors and associated methodsfor diagnostic measuring or testing using the principle of a phase orfrequency shift from an initial resonance point in response to exposureof the sensors to certain materials.

BACKGROUND OF THE INVENTION

There are a variety of instruments and measurement techniques fordiagnostic testing of materials related to human health, veterinarymedical, environmental, biohazard, bioterrorism, agricultural commodityand food safety. Still, a solution for diagnostic testing and analysisof chemical or biological materials at the point of need remainslimited. Diagnostic testing traditionally requires long response timesto obtain meaningful data, involves expensive remote or cumbersomelaboratory equipment that costs thousands of dollars located in acentralized laboratory, requires large sample sizes, utilizes multiplereagents, demands highly trained users, may require numerous steps,and/or involves significant direct and indirect costs. For instance, inboth the veterinary and human diagnostic markets, most tests requirethat a specimen be collected from the patient and sent to thelaboratory, but the results are not available for several hours or dayslater. As a result, the patient may leave the caregiver's office withoutconfirmation of the diagnosis and the opportunity to begin immediatetreatment.

Other problems related to portable devices include diagnostic resultsthat are limited in sensitivity and reproducibility compared toin-laboratory testing. Fast response times are desirable and oftencritical to the identification of chemical and/or biological materials,such as in providing timely medical attention or in averting the spreador exposure of public health threats. Direct costs relate to the labor,procedures, and equipment required for each type of analysis. Indirectcosts partially accrue from the delay time before actionable informationcan be obtained, e.g., in medical analyses or in the monitoring ofchemical processes. Many experts believe that the simultaneous diagnosisand treatment enabled by an effective point of need diagnostic testingsystem would yield clinical, economic and social benefits.

Biosensors based on piezoelectric properties of materials have been usedin detecting very small quantities of materials. Piezoelectricresonators used as sensors in such applications are sometimes called“micro-balances.” A piezoelectric resonator is typically constructed asa thin planar layer of crystalline piezoelectric material sandwichedbetween two electrode layers. When used as a sensor, the resonator iscoated with a binding layer which, when exposed to the material beingdetected, allows the material to bind to the surface of the resonator.Modern resonators are fabricated using MEMS techniques and can beconstructed to be so small that their resonant frequency is on thegigahertz scale. In general, resonators having higher resonantfrequencies are more sensitive.

The conventional way of detecting the amount of the material bound onthe surface of a sensing resonator is to operate the resonator as anoscillator at its resonant frequency. As the material being detectedbinds on the resonator surface, the mass of the resonator increases andthe resonant frequency of oscillation is consequently reduced. Thechange in the resonant frequency of the resonator over time, presumablycaused by the binding of the material on the resonator surface, isindicative of the amount of the material that is bound on the resonatoror the rate at which the material accumulates on the resonator surface.From this data, a concentration of the material of interest, or analyte,present in the sample can be computed.

Conventionally, biosensors of this type generally include an assembly inwhich an intrinsic biosensor is surrounded by at least one fluidicchannel, which is coupled to a sample reservoir for presenting a sampleto the biosensor in a controlled manner. Most conventional biosensorconfigurations also include a mechanism for controlling and maintaininga desired temperature of the sample as it is presented to the intrinsicbiosensor. In operation, the sample is drawn through the fluidic channeland across the intrinsic biosensor by application of vacuum or similaractuation pressure at a vacuum port. Oftentimes, the sample must berefined prior to introduction into the assembly by the addition ofbuffer or removal of certain elements such as whole cells or otherparticulates, which can interfere with the accuracy of the measurements.This refining step can be cumbersome and costly, making suchmeasurements impractical for field applications. In addition, theintroduction of the sample changes the physical environment in which themeasurement is made. For example, the resonator oscillates at differentresonant frequencies when exposed to a liquid sample versus prior toexposure when the resonator is in free air oscillation due todifferences in viscosity between the two fluids.

Another approach involves stabilizing the measurement environment priorto introduction of the sample reagent. For instance, one type ofinstrument has two separate fluid reservoirs in which the firstreservoir contains a buffer solution and the second reservoir containsthe sample reagent. In operation, the buffer solution is introduced tothe biosensor first, and the system is allowed to stabilize. Next, avalve switches to the sample reagent and measurements are made. Theintroduction of the buffer solution permits the sensor's oscillation tobe tuned, or adjusted in accordance with the viscosity and temperatureconditions of the buffer, which closely approximates the viscosity andtemperature of the sample. The objective of this tuning is to operatethe resonator as close as possible to its ideal resonant frequency formaximum sensitivity. One drawback of this approach is the introducedcomplexities and processing time requirements, which can result inexpensive, error-prone, and time-consuming test results.

Another challenge faced by designers of resonant biosensors is in makingquantifiable concentration measurements on highly concentrated samples.One trade-off of having the increased sensitivity of micro-scale (orsmaller) resonators is their susceptibility to becoming rapidlysaturated with analyte. In certain tests, a sensor can become saturatedin a matter of seconds or even in a fraction of a second. In this case,although the sensor can operate as a simple detector of the presence ofanalyte, it cannot accurately measure the rate at which the analytebinds to the sensor, commonly referred to as binding kinetics.Measurement of binding kinetics is needed for quantification of theconcentration of the analyte, can be impossible to obtain with anysuitable accuracy or repeatability in highly concentrated samples usingconventional techniques. Moreover, when analyte begins to bind to theresonator in significant quantity immediately from the moment that theresonant sensor is introduced to the sample, the instrument may not havethe time to achieve its required stabilization prior to the taking ofmeasurements, or to perform tuning of the resonator either before orafter the taking of measurements, thereby further exacerbating theproblem.

In view of the above, a practical solution is needed to enable themeasurement of binding kinetics in sensitive instruments, particularlyearly binding kinetics from the time the sample is introduced to thesensor. Additionally, it would be desirable to perform such measurementswithout having to undertake the complexity of stabilizing the resonatingsensor using a buffer solution or specially-refined sample.

SUMMARY OF THE INVENTION

One aspect of the invention is directed to detecting a presence of asubject material in a fluid sample using at least one resonating sensorimmersible in the fluid sample. Binding kinetics of an interaction of ananalyte material present in the fluid sample are measured with theresonating sensor, which has binding sites for the analyte material.Prior to exposing the resonating sensor to the fluid sample, operationof the resonating sensor is initiated, which produces a sensor outputsignal representing a resonance characteristic of the resonating sensor.Optionally, a reference resonator is used that produces a referenceoutput signal. The reference resonator lacks binding sites for theanalyte. Introduction of a fluid sample to the resonating sensor isautomatically detected based on detection of a characteristic change inthe sensor output signal or a reference output signal, or both. Inresponse to the detecting of the introduction of the fluid sample,automated measurement of the binding kinetics of the analyte material tothe resonating sensor are measured.

According to another aspect of the invention, an apparatus for measuringbinding kinetics of an interaction of an analyte material present in afluid sample is provided. The apparatus includes a resonator interfaceadapted be operatively coupled with one or more resonating devices, atleast one of which is a sensing resonator having binding sites for theanalyte material. The one or more resonating devices are adapted to bedriven into an oscillating motion by actuation circuitry. Measurementcircuitry is arranged to be coupled to the one or more resonatingdevices via the resonator interface. The measurement circuitry isconfigured to measure one or more resonator output signals representinga resonance characteristic of the oscillating motion of the one or moreresonating devices. A controller is operatively coupled with theactuation and measurement circuitry, and configured to detectintroduction of the fluid sample into contact with the one or moreresonating devices based on detection of a characteristic change in theone or more resonator output signals; and in response to the detectionof the introduction of the fluid sample, initiate measurement of thebinding kinetics of the analyte material to the at least one sensingresonator.

A method for measuring binding kinetics of an interaction of an analytematerial present in a fluid sample with one or more resonating devices,at least one of which is a sensing resonator having binding sites forthe analyte material, is provided in another aspect of the invention.According to this method, prior to exposing the one or more resonatingdevices to the fluid sample, operation of the one or more resonatingdevices is initiated that produces one or more resonator output signalsrepresenting a resonance characteristic of each of the one or more ofthe resonating sensors. Introduction of a fluid sample to the one ormore resonating devices is automatically detected based on detection ofa characteristic change in the one or more resonator output signals,such as resonance frequency or phase angle, for example. In response tothe detecting of the introduction of the fluid sample, automatedmeasurement of the binding kinetics of the analyte material to the atleast one resonating sensor having the binding sites is initiated.

The measurement of the binding kinetics can be based on change of aresonance frequency of the one or more resonating devices, which can bea differential mode measurement in some embodiments. The bindingkinetics can be based on a total change over time, or on a rate ofchange measurement.

A number of advantages of the invention will become apparent to personsof skill in the relevant art based on the following description of thepreferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be more completely understood in consideration of thefollowing detailed description of various embodiments of the inventionin connection with the accompanying drawings, in which:

FIG. 1A is a diagram illustrating a hand-held resonance shift detectorsystem according to one embodiment.

FIG. 1B is a diagram illustrating the hand-held resonance shift detectorsystem of FIG. 1A with the sensor detached from the interconnector andthe interconnector attached from the instrument.

FIG. 2 is a diagram illustrating a laboratory bench resonance shiftdetector system according to one embodiment.

FIG. 3 is a block diagram view of an exemplary implementation of aresonance shift detector system that uses phase shift detectionaccording to one embodiment.

FIG. 4 is a block diagram view of an exemplary implementation of aresonance shift detector system that uses phase shift detectionutilizing a one-port sensing resonator and a one-port referenceresonator according to one embodiment.

FIG. 5 is a block diagram illustrating an exemplary implementation of aresonance shift detector system that uses frequency shift detectionaccording to one embodiment.

FIG. 6A is a schematic top view of a resonator assembly according to oneembodiment.

FIG. 6B is a schematic of a partial layer view of the resonator assemblyof FIG. 6A.

FIG. 7 is a diagram illustrating a frequency ladder approach ofdetermining frequency divisions for operating multiple sensor/referenceresonator pairs at non-interfering frequencies in a multiplexedembodiment.

FIG. 8A is a schematic diagram illustrating a simplified model of aresonator in the resonance shift detector system according to oneembodiment.

FIG. 8B is a schematic diagram illustrating a more detailed model of aresonator sensor in the resonance shift detector system according to oneembodiment.

FIG. 9A is a diagram illustrating a multi-stage frequency sweeptechnique for in-situ tuning of a resonator according to one embodiment.

FIG. 9B is a diagram illustrating a practical implementation of thetechnique of FIG. 9A according to one embodiment.

FIGS. 10A and 10B are graphs depicting evolution of biosensor signalsover time during system operation for a frequency shift detectionbiosensor and a phase shift detection biosensor, respectively, accordingto embodiments of the invention.

FIGS. 11A and 11B are a flow diagrams illustrating an exemplaryoperation, respectively, of systems that control and monitor thefrequency shift detection biosensor, and the phase shift detectionbiosensor, the outputs of which are represented in FIGS. 10A and 10Babove according to embodiments of the invention.

While the invention is amenable to various modifications and alternativeforms, specifics thereof have been shown by way of example in thedrawings and will be described in detail. It should be understood,however, that the intention is not to limit the invention to theparticular embodiments described. On the contrary, the intention is tocover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention as defined by the appended claims.

DETAILED DESCRIPTION

While this invention may be embodied in many different forms, there aredescribed in detail herein specific preferred embodiments of theinvention. This description is an exemplification of the principles ofthe invention and is not intended to limit the invention to theparticular embodiments illustrated.

One aspect of the present invention is directed toward a simple,effective, cost-efficient, reliable, repeatable, sensor for resonanceshift detection of chemical and/or biological materials that compensatesfor the variation of the unique resonant frequency in individualresonators. For the sake of brevity, these devices are referred toherein as biosensors, though it should be understood that they can beused to detect materials in samples other than biological samples.Resonance shift detection, in various embodiments, can be based on phaseshift or frequency shift. One type of embodiment includes a sensor andresonance shift detection system that can compensate or adjust for thevariation in individual resonator frequency and environmental elementsthat can influence the operation of a resonator at its resonantfrequency, while providing cost-efficient, reliable, repeatable andaccurate results. In some embodiments of the invention, the resonanceshift detection system includes systems and methods that avoid thecomplications created by a pair of resonators that are not identicallymatched by operating each resonator at its own, potentially unique,resonant frequency. By operating each resonator at or near its idealresonance, the benefits of increased sensitivity can be realized. Thisin turn, provides for faster and more accurate testing.

In some embodiments, multiple sensor-mounted resonators are separately,or individually, driven at different resonant frequencies. Further, aseparate phase or frequency offset measurement is performed for eachresonator. Each offset is then compared to the other resonators' phaseor frequency offsets. In another embodiment the phase or frequencyoffset is measured relative to the driving signal provided to eachrespective resonator and not relative to the other resonators' phase orfrequency offsets.

In some embodiments, a sensor for resonance shift detection of chemicaland/or biological materials includes one or more printed circuit boards,with one or more resonator dies mounted thereon. The resonator dies inone example embodiment are bulk acoustic wave (BAW) devices. The closeproximity of two or more resonators ensures that the resonators aresubjected to substantially identical environmental conditions during amaterial sensing operation. The use of sensor/reference pairs in whichone resonator is a sensor and the other resonator is a referenceresonator, effectively allows accurate resonance shift measurements andcancellation of environmental effects during material sensing operationsusing the sensing assembly.

According to various implementations, the sensors for resonance shiftdetection can include back-to-back PCB configurations utilizing twosubstantially different PCBs. In one approach, the resonator on one PCBis situated off-center while the resonator on the other PCB is centered.In this configuration, the reference and sensing resonators can stillhave sufficient distance there-between to reduce cross talk between thetwo resonators. In another aspect of the present invention, theresonators on the two PCBs are constructed such that the back-to-backPCB configuration results in the reference and sensing resonators beingdirectly opposed.

In some embodiments, the sensing resonator is coated with a differentmaterial than a reference resonator depending upon the material to bedetected. By varying the coating on the resonators, the resonance shiftdetection system allows universal use for various diagnostic testing ofchemical and/or biological materials without changing any of the othersystem structural components. Sensors for resonance shift detection ofchemical and/or biological materials effectively allow fast responsetimes for the detection of the respective chemical and/or biologicalmaterial, in the field detection capabilities, small sample sizes,minimally trained individuals, low direct and indirect costs, andelectronically transmittable data.

In some embodiments, the resonance detection system includes an on-boardpower source, such as a battery, super capacitor, solar-powerarrangement or any combination thereof, for powering the electronicinstrument, and an easily mounted disposable sensor that may becontained within a sensor housing assembly. The sensor has a biologicalcoating that specifically binds with the desired target molecule andprovides the mechanism of detection and quantification. A specimen(whole blood, urine, saliva, or any other liquid) is drawn into thesensor housing assembly and brought into contact with the resonators ofthe sensor, which may or may not be a single-use sensor, within thesensor housing assembly. Electrical measurements of the sensor (i.e. achange in phase or resonant frequency of the RF wave) indicate if thetarget is present, and if present, its concentration. For each analysis,a new sensor is attached to the instrument.

In some embodiments, the tuning of the sensor can be performed todetermine the exact resonant frequency of the resonator prior to thematerial sensing operation. This tuning can account for the variouselectrical properties associated with the sensors' physical connectionto an instrument as well as the immediate environmental conditions. Asecond tuning can also optionally be performed in situ promptly afterthe sensor is introduced into a specimen for sensing.

In general, the rate of change of the resonance characteristics varieswith the concentration of the target. The rate of change of theresonance characteristics for the most concentrated sample occurs fasterthan the lowest concentrated sample. The binding kinetics, such as, forinstance, the slope of the response of known samples, or a totalmagnitude of a change in the response to introduction of the sample as afunction of time, can then be used to generate a calibration curve sothat the concentration of unknown samples can be determined byinterpolation of the sensor response against the curve. Duringmanufacturing, sensors may be factory calibrated by production lot,allowing the user easy determination of the concentration of an unknownsample without further calibration.

Still another aspect of the invention is directed to fast in-situ sensortuning, which allows the driving signal frequency to be determined veryquickly from the time a sensor is exposed to the sample under test.

In another aspect of the invention, a resonating sensor system isconfigured to deliver accurate test results even with directintroduction of unrefined sample onto a resonating sensor's surface. Thebiosensor generates a real-time electrical output signal proportional tothe binding of analyte onto the sensor's surface. The rate of analytebinding, referred to as binding kinetics, is proportional to theconcentration of analyte in the unrefined sample. In this aspect, theintroduction of the sample to the surface of the resonating sensor isautomatically detected as a step change in the sensor's resonancecharacteristics. By very rapidly initiating measurement in response tothe detection of the sample introduction, the initial on rate of thebinding kinetics can be measured. Likewise, for total changemeasurement, having a precise starting point corresponding to actualintroduction of the sample to the sensor facilitates accuratemeasurement of the binding kinetics.

In a related aspect of the invention, as applied to systems utilizingdriving signals having a set frequency such as phase shift detectionsystems, in order for the biosensor system to deliver accurate testresults, a fast tuning is be performed at the initial time that theunrefined sample is presented to the sensor. Some of the embodimentsdescribed below accomplish this with an initial air tuning prior tosample introduction, nearly instantaneous electronic detection of samplepresentation to sensor's surface, followed by fast tuning performed inthe presence of the sample to be analyzed.

Turning now to the drawings, the components of the resonance shiftdetector system according to some embodiments of the present inventionare illustrated. In some embodiments, the resonance shift detectorsystem can be relatively small in size to be portable such that it canbe utilized in the field for specific diagnostic testing applications.In some other embodiments, the resonance shift detector system can beconfigured for diagnostic testing in a laboratory setting. As shown inFIGS. 1A and 1B resonance shift detector system 10 is illustrated in ahand-held or portable configuration that includes an instrument 12 acapable of being interfaced with a sensor assembly 30 by aninterconnector 20, which can be used for point of need diagnostictesting in the field.

As illustrated in FIG. 2, the resonance shift detector system isillustrated in a laboratory bench or more permanent configuration thatincludes an instrument 12 b, such as a Network Analyzer, capable ofbeing interfaced with a sensor assembly 30 by an interconnector 20. Thesensor assembly 30 mounted on an interconnector 20 and coupled to alaboratory-bench instrument 12 b, such as a Network Analyzer, allowsdiagnostic testing in a laboratory setting, quality control testing of abatch of sensors during production, and/or the development of coatingson the sensor assembly 30 for target material diagnostic testing. Theinstrument including, but not limited to hand-held instrument 12 a andlaboratory-bench instrument 12 b, may have means for connection to theinternet or otherwise transferring information, such as one or more USBports, wireless connection, or the like.

In some embodiments, the interconnector 20 can contain a data storagedevice such as a ROM or flash EEPROM. The data storage device may serveto set up the instrument for specific market applications by includingsoftware or identification information that allows the instrument tounderstand the particular use of the resonance shift detector system 10as it relates to the sensor assembly 30. For instance, the read-onlymemory may contain basic information or algorithmic instructions for theinterpretive logic of the instrument that relates to the output signalof the sensor assembly 30, which may serve to limit the resonance shiftdetector system 10 to specific applications, such as limited only to usein one of: veterinary applications, toxicology applications; drugs ofabuse applications; GMO grain applications, for example.

The data storage device can also contain sensor-type specificinformation such as the general frequency range or approximate resonancefrequency of the resonator as determined during post-production testing.This information could, for example, reduce sensor detection andcalibration setup time when a new sensor is coupled to an instrument. Ina related embodiment, the data storage device contains lookup tables ofcalibration correction constants that are indexed by lookup codesindividually determined for the sensors at the factory. In various otherembodiments, the lookup code may be supplied via printed label, barcodelabel, or using a RFID tag.

In another related embodiment, the sensor includes a read-only memory(ROM) or small flash device having its own specific calibrationconstants specific to the individual sensor assembly 30. This data couldbe supplied based on factory calibration performed on a representativesample taken from the manufactured lot in which the individual sensorassembly 30 was fabricated. In yet another embodiment, the instrument isconfigured with a network interface device and associatedfirmware/drivers, which enable the device to automatically initiate aquery over a network to obtain calibration constants for the specificsensor. This embodiment eliminates the need for maintaining calibrationdata locally. Instead, when a new sensor is attached, the instrumentdetermines the serial number associated with the particular sensor(using RFID, bar code scanning, etc.), and uses that information to formits query. The database having specific sensor calibration data may bestored on a server located at the laboratory facility, or remotely(e.g., at the manufacturer's facility), in which case the network overwhich the query is placed is a wide area network (WAN) such as theInternet.

Sensor assembly 30 includes one or more resonators such as bulk acousticwave devices, for instance, described in greater detail below. Invarious embodiments, sensor assembly 30 may or may not include circuitrythat interfaces with the one or more resonators. For instance, in onetype of embodiment, actuation circuitry that causes the one or moreresonators to oscillate, is incorporated into the sensor assembly 30. Inanother embodiment of this type, measurement circuitry, includinganalog-to-digital conversion, is incorporated into sensor assembly 30.In another type of embodiment, the actuation and measurement circuitryis located in the housing of instrument 12 a or 12 b.

In one embodiment of the invention, as depicted in FIG. 3, a system thatdetects resonant characteristics changes of a sensing resonator and areference resonator. In this example embodiment, the phase angle of eachresonator is the resonant characteristic that is being monitored.

A sample resonator 44 and reference resonator 54 are each coupled toseparate directional couplers 52. The directional couplers 52 providetheir respective resonators 44, 54 with a signal generated by amicrocontroller 55 controlled frequency synthesizer 56 and a variablegain amplifier 58. The directional couplers 52 also each output theincident signal and the reflected sensor signal 35 to a vector signaldetector 59. The vector signal detector 59 for each resonator processesthe signals to produce an output signal indicative of a phase differencebetween the input and output frequency signals. These signals aredelivered to an analog to digital converter and then read by themicrocontroller 55.

In various embodiments, microcontroller 55 uses the output signal foreither one, or both, of the sensing and reference resonator to detect anabrupt change indicative of introduction of the sample to theresonators, as will be described in greater detail below.

Additionally, microcontroller 55 is programmed to compare any differencein phase change, or the rate of phase change over time, between theincident signal and the reflected sensor signal 35 of both the sampleand reference resonators. Because the change in phase of the sampleresonator 44 is caused mainly by the binding of the material beingdetected on the surface of the sensing resonator, a greater phase changeover time will be observed in comparison to any phase change over time,due to environmental effects, observed at the reference resonator.Because of the potential effect temperature can have on the resonantfrequency of the resonators, a temperature sensor 57 can be included toprovide temperature data to microcontroller 55.

According to one aspect of the present invention, separate sensing andreference resonators are independently driven at their own uniqueindividual resonant frequencies, which are separately determined. Theuse of distinct driving frequencies provides improved sensitivity sincethe sensor and reference resonators are operated their respective exactresonance frequencies (or as close to them as feasible in a givensystem), and any changes to the resonant frequency is more easilydiscernable.

FIG. 4 illustrates an example measurement arrangement according to oneembodiment, using a one-port sensing resonator 44 and a one-portreference resonator 54, each of which receives its own separate drivingsignal. A one-port resonator has an electrode 46, 48 that is used forboth signal input and output. The other electrode 49, 50 of eachone-port resonator is typically grounded.

This embodiment includes a signal source 21, which includes avoltage-controlled oscillator 60 and a multiplexer 62, provides an inputsignal of a first frequency which is at or near the resonant bands ofthe first resonator's resonant frequency. Signal source 21 also providesan input signal of a second frequency which is at or near the resonantbands of the second resonator's resonant frequency. In a variation ofthe arrangement illustrated in FIG. 4, a separate oscillator, such as anadditional VCO (not shown) can provide an input signal of a frequencyfor each of the respective resonators, which could eliminate the needfor multiplexer 62.

The input signal provided by the signal source 21 is directed bydirectional couplers 23, 24 to their respective sensing and referenceresonators 44, 54. The reflected output signals of the resonators 44, 54are directed to the phase detectors 25, 26 by the respective couplers23, 24. Each of the phase detectors 25, 26 additionally receives areference signal from a corresponding internal reference signalgenerator 27, 28. Phase detectors 25 and 26 process the sensor andreference signals to produce output signals indicative of a phasedifference between the input and output frequency signals of theirrespective resonators. The output of phase detectors 25,26 is selectedvia multiplexer 63 (which operates generally synchronously withmultiplexer 62—both may be implemented in a single package with a commoncontrol input, or may be separate devices, each with its own controlinput), and converted to a digital representation by analog-to-digitalconverter (ADC) 64, which is interfaced with controller 55.

Controller 55 is programmed to apply logic to interpret the outputsignals. Introduction of the sample to the resonators generally causes acommon abrupt change to both, the sensing and reference resonators'output signals, which controller 55 is programmed to detect. The bindingof the material being detected on the surface of the sensing resonator44 generally causes a change of phase angle dΦ/dt between the sensingresonator and reference resonator when the detected material is present.

The phase detectors 25, 26 in the illustrated embodiment can include adouble-balanced mixer (or a mathematic multiplier) which receives thesensor and reference signals. The output of the mixer is passed througha low-pass filter which eliminates a time dependent term and leaves onlythe DC term as the output of the phase detector 25. As provided in moredetail in U.S. Pat. No. 5,932,953, the disclosure of which isincorporated by reference herein, the resulting measured phase shiftchange can be used to derive the total amount of the material bound onthe surface of the sensing resonator 44. In some embodiments, the signalsource 21 generates an analog signal and the phase detector 25 generateseither an analog or a digital output signal after receiving the signaland reference signals and processing the information there from.

In the embodiment illustrated, the signal from each respective resonator44, 54 is directed to a separate respective phase detector 25, 26 eachrespective phase detector 25, 26 processing the respective sensor orreference signal to produce a phase signal indicative of a phase shift.The phase shift data is converted to digital data that can then comparedto each other, or to their respective source frequencies, by thecontroller 55 to determine the net difference in phase shift between thesensing and reference resonators. The different phase shift being causedby the binding of the material being detected on the surface of thesensing resonator 44 and not on reference resonator 54. Controller 55can be configured to periodically sample the output of phase detectors25, 26 or any other appropriate mechanism for observing and recordingthe change in phase of the two signals during operation.

The sensing resonator 44 is coated with a test reagent that binds to orcaptures the analyte to be detected during the diagnostic testing. Thereference resonator 54 is coated with a reference reagent that does notbind with or otherwise capture the analyte during the diagnostictesting.

FIG. 5 is a block diagram illustrating a measurement arrangement with atest and reference resonator according to another embodiment in which afrequency shift detection scheme is used to detect a change inresonance. Test resonator 70 and reference resonator 71 are connectedrespectively to amplifiers 76 and 77 as shown. Amplifiers 76, 77 areconstructed such that, when operated with resonators 70, 71, theygenerate output signals at the resonant frequency of resonators 70, 71.Thus, changes in the resonant frequency of test resonator 70 orreference resonator 71 (such as those induced by the capture of analyteon the surface of the test resonator, or by localized environmentalchanges in the sample solution in which the test and referenceresonators 70, 71 are immersed) will cause changes in the resonantfrequency delivered from amplifiers 76, 77 to digital counters 78, 79.

The resonant frequencies are generated by amplifiers 76 and 77, andpassed to digital frequency counters 78 and 79, the outputs of which areinterfaced with controller 80. Controller 80 thus has a continuousreading of each resonant frequency, the changes in which representchanges in the resonance of test resonator 70 and reference resonator71. As will be described in greater detail below, in one embodiment,controller 80 is programmed to detect an abrupt change in resonancecharacteristic of either one, or both, the sensing and referenceresonator, as an indicator of introduction of the sample. Controller 80is further programmed to ascertain differences in the changes ofresonance characteristics between test resonator 70 and referenceresonator 71, along with the rate of change in resonance of thisdifference. From this information, controller 80 is programmed todetermine whether the test resonator 70 has experienced binding ofanalyte and, from the rate of change in its resonance characteristic(relative to that of the reference resonator 71), to determine theconcentration of analyte in the sample fluid.

During the sampling process, the sensor assembly 30 is introduced into aliquid or gaseous sample, or the sample aliquot may be introduced to thesensing and reference resonators 44, 54 by way of a sensor housingassembly. The liquid sample for the diagnostic test may include blood,urine, serum, saliva, water, or any other liquid sample that may be ofinterest. As soon as the sample contacts the sensing and referenceresonators 44, 54, there is a change in signal from the resonators 44,54. The instrument 12 is waiting to receive the change in signal, andonce the change in signal is detected, the instrument 12 begins theinterpretative sequence of collecting data. The instrument 12 continuesto collect data until either (i) the instrument 12 times out because thesignal has not changed, or (ii) depending upon the speed with which thesignal changes, the instrument 12 will stop collecting data once enoughdata is received to give an interpretation of the diagnostic test. Aninterpretation of the diagnostic test may include an indication that thetarget material, or analyte, has been bound or captured onto the sensingresonator and a quantification of the target material.

Referring now to FIG. 6A the top surface of an example resonatorassembly is illustrated, which represents the sensing resonator assemblyand reference resonator assembly. For ease of reference, the followingdescription refers to the sensing resonator assembly 44, although thedescription is equally applicable to the reference resonator assembly54. The top surface of the sensing resonator assembly 44 contains a setof solder bumps 45 a-45 d. Solder bumps 45 a-45 c are connected toground within the resonator assembly 44. Solder bump 45 d is connectedto the resonator 45A by way of a via through the piezoelectric layer anda resonator conductor between, which is further illustrated by thepartial layer view in FIG. 6B. The resonator assembly 44 is alsocantilevered over the edge of the printed circuit board to allow theresonator 45A to be exposed to the surrounding environment during thetesting process. In some embodiments, the solder pads may be on adifferent side of the resonator assembly 44 than the resonator 45A, suchthat the resonator assembly 44 does not necessarily have a cantileveredconfiguration. In yet other embodiments, the PCB may be configured suchthat the resonator assembly is mounted in a depression in the PCB andthe electrical connection between the resonator assembly and PCB isaccomplished with a conductor such as a conducting epoxy.

In the construction of resonators, the relative surface area of theresonator 44A is directly related to the frequency at which theresonator resonates, with higher frequency resonators having a smallersurface area and lower frequency resonators having a larger surfacearea. For example, a smaller-sized resonator (e.g. diameter of 154.4 μm)has a resonant frequency of 2.25 GHz while a larger-sized resonator(diameter of 254 μm) has a resonant frequency of 900 MHz. Accordingly,it is contemplated that resonators with various resonant frequencies maybe used depending upon the desired resonant frequency and any regulatoryrestrictions on the frequencies available to be used. The thickness ofthe piezoelectric layer also affects frequency with a thinnerpiezoelectric resonating at a higher frequency than a thicker layer. Asdiscussed previously, while various resonators of similar size havegenerally similar resonant frequencies the slight variation in size,potentially due to variances in manufacturing, can result in similarresonators with close but meaningfully different resonant frequencies.

In some embodiments, a back-to-back paddle configuration allows thesensing resonator 44 and the reference resonator 54 to be located in aclose proximity with each other, the two resonators are subjected tosubstantially identical environmental conditions during a materialsensing operation, which allows for accurate resonance shiftmeasurements and effective cancellation of the environmental effects.Environmental effects that may be cancelled may be a result ofviscosity, pH, temperature, particulates, and any other environmentconditions within the sample that will affect the sensing resonator 44during diagnostic testing.

In another aspect of the invention, multiple sensor/reference resonatorpairs are employed. Each sensor/resonator pair may be configured todetect a different material, or multiple ones of sensor/resonator pairsmay be used in order to provide redundantly for improved accuracy orreliability of the instrument. In related embodiments, provisions aremade to reduce cross-talk between the multiple pairs of sensor/referenceresonator pairs.

One approach is to provide physical separation between pairs. Physicalseparation may be achieved by placing the different sensor/referenceresonator pairs on separate substrates to provide mechanical andelectrical isolation. Another way of achieving physical separation is byplacing the different sensor/reference pairs far enough apart so thatany mechanical or electrical coupling is rendered nominal.

Cross-talk between the resonators may also be reduced or eliminated bythe pairs of sensor/reference resonator pairs being operated atdifferent frequencies. For example, two or more pairs of resonatorsmounted on a single PCB can be fabricated to have their nominal resonantfrequencies spaced apart by an amount sufficient to reduce anycross-talk to a negligible level. The spectral spacing sufficient toachieve the isolation depends on a variety of factors, such as thequality factor of the oscillation which, in turn, depends on thematerials, construction, and geometry of the resonators themselves;additionally, the required spectral spacing to reduce crosstalk to anacceptable level can depend on the selectivity of the measuringcircuitry. The frequency separation can be defined in terms ofpercentage of driving frequency. For instance, the separation can beabout 1-5% of driving frequency. Thus, in embodiments where the resonantfrequency is in the range of 750-1000 MHz, the frequency separation maybe approximately 15 MHz between the multiple sensor/reference resonatorpairs.

FIG. 7 is a diagram illustrating a process of determining resonantfrequencies for different pairs of resonator on a multi-resonator boardaccording to one embodiment. The approach of this embodiment uses afixed frequency (DeltaF) Ladder design. In a multiplexed design of aresonator group, the frequency difference (DeltaF) between any tworesonators at the adjacent ladder steps is such that it reduces thecross talk between the resonators to a negligible amount. The frequencydifference between the adjacent ladder steps can be fixed or may bevariable and is defined by the application or test. As depicted in FIG.7, each sensor/reference resonator pair is operated at a similarfrequency. However, other sensor/reference resonator pairs are eachoperated at a frequency difference DeltaF.

In another type of embodiment, groups of more than two sensors may beutilized in a measurement arrangement. Rather than being arranged inpairs, groups may be composed on three or more sensors, of which thereis more than one sensing resonator, and/or more than one referenceresonator. In one type of configuration, a group of resonators isarranged in close proximity so that the group can be exposed to the sametest environment. One such group of sensors can include a total of eightresonators, of which seven are sensors, and one is a reference. As avariation of this example, embodiment, a different group of eightresonators includes six sensors and two reference resonators.

In operation, for each group of resonators, the resonance shift of eachsensing resonator can each be compared against a certain individualreference resonator. Alternatively, an average (or other statisticallyaggregated) resonance shift of more than one sensor can be compared withan average (or otherwise aggregated) resonance shift of more than onereference resonator of the group. In various instruments implementingaspects of the invention, a variety of different measurementarrangements using groups of more than two sensor/reference resonatorscan be employed in order to achieve improved accuracy or precision, orto provide a more comprehensive test which can detect a plurality ofdifferent materials in the sample under test.

Various embodiments, including one or more resonators mounted on aplurality of connected printed circuit boards, are also contemplated.One such embodiment includes a sensor with multiple PCB layers, eachlayer or sub-group of layers including one or more resonators mounted ina staggered or grid layout across the multiple PCB layers, eachresonator having the same axis of resonance. In other embodiments,multiple individual sensors with one or more resonators are constructedin any of various configurations including, but not limited to a cross,diamond, triangle, square, pentagonal or circular orientations formedfrom three or more stacked PCB layers. An appropriate interconnector canbe employed to couple the plurality of PCB layers to an instrumentconfigured to operate a multi-resonator sensor.

FIG. 8A is a circuit diagram illustrating a simplified resonator sensormodel 40. The model 40 provides a link between the physical property ofthe resonator crystal and the oscillator. The physical constants of thecrystal determine the equivalent values of R1, C1, L1 and C0 of thedevice. Resistance (R1) is a result of bulk losses, C1 is the motionalcapacitance, L1 is the motion inductance as determined by the mass, andstatic capacitance (C0) is made up of the electrodes, the holder, andthe leads. When operated far off resonance the structure is simply acapacitor with capacitance C0, but at the precise resonant frequency ofthe crystal the circuit becomes a capacitor and resistor in parallel.The reactance of the crystal approaches zero at the point of seriesresonance and reaches a maximum at the anti-resonant frequency fA.

-   -   One example of determining the various properties of the        resonator is a lumped element method including the following        steps performed on data gathered in a SnP 1-port data file:    -   1) Convert s11(f) to z11(f) for all frequency measured.    -   2) The real (z11) at the highest frequency (fh) is the contact        resistance Rc=real (z11(fh)).    -   3) The stray capacitance Co is also computed at the highest        frequency (fh)

Co=1/[2*pi*fh(hz)*imag(z11(fh))]

-   -   4) Remove the effects of Rc by subtracting it from z11.        z11′(f)=z11(f)-Rc    -   5) Series resonant frequency fs can be determined when the sign        of Imag[z11″(f)] changes neg to pos.    -   6) Motional inductance is computed as

Ls=(1/(4*pi))*(imag[z11″fs)]−imag[z11″(fs−Δf)]/Δf where Δf=500 kHz

-   -   7) Motional resistance is Rs=real[z11″(fs)]    -   8) Motional capacitance is Cs=1/[(2*pi*fs)̂2*Ls]

FIG. 8B is a diagram illustrating a more sophisticated resonator model42 that includes contact resistance (Rc). Both the contact resistance Rcand the static capacitance C0 are important variables that should bede-embedded in order to observe the changes happening on the resonatorsurface. As will be understood by those skilled in the art, theinterconnector 20 may also introduce a translation and rotation effecton the measurements of the resonator sensors. Because the act ofmounting a crystal on a sensor can affect the contact resistance Rc andthe static capacitance C0 of the resonator it is important that theseparameters be accounted for in the calibration, tuning, and use of theresonator sensors.

One example method of determining the various properties of theresonator with C0 removed includes the following steps:

-   -   1) Convert s11(f) to z11(f) for all frequency measured.    -   2) The real (z11) at the highest frequency (fh) is the contact        resistance Rc=real (z11(fh)).    -   3) The stray capacitance Co is also computed at the highest        frequency (fh)

Co=1/[2*pi*fh(hz)*imag(z11(fh))]

-   -   4) Remove the effects of Rc by subtracting it from z11.        z11′(f)=z11(f)−Rc    -   5) Remove the effects of Co by removing it from

y11′(f) or z11″(f)=1/[(1/z11′(f))−j(2*pi*f*Co)]

-   -   6) Series resonant frequency fs can be determined when the sign        of Imag[z11″(f)] changes from neg to pos.    -   7) Motional inductance is computed as

Ls=(1/(4*pi))*(imag[z11″(fs)]−imag[z11″(fs−Δ)])/Δf where Δf=500 kHz

-   -   8) Motional resistance is Rs=real[z11″(fs)]    -   9) Motional capacitance is Cs=1/[(2*pi*fs)̂2*Ls]

The above steps can in a third example be modified to result in thefollowing method of modeling a resonator:

-   -   1) Convert s11(f) to z11(f) for all frequency measured.    -   2) The real (z11) at the highest frequency (fh) is the contact        resistance

Rc=real(z11(fh)).

-   -   3) The stray capacitance Co is also computed at the highest        frequency (fh),

Co=1/[2*pi*fh(hz)*imag(z11(fh))]

-   -   4) Remove the effects of Rc by subtracting it from z11.

z11′(f)=z11(f)−Rc

-   -   5) Motional inductance is computed as

Ls=(1/(4*pi))*(imag[z11′(fs)]−imag[z11′(fs−Δf)])/Δf where Δf=500 kHz

-   -   6) Motional resistance is Rs=real[z11′(fs)]    -   7) Motional capacitance is Cs=1/[(2*pi*fs)̂2*Ls]        The resonator models can be utilized to screen manufactured        resonators in order to evaluate and select appropriate        resonators.

In one embodiment, the following method can be used to evaluateresonators:

-   -   1) Load S1p files for all resonators.    -   2) Convert s11(f) to Z11(f) for all frequencies measured.    -   3) Compute Contact Resistance at highest frequency (fh)

Rc=real(z11(fh))

-   -   4) Remove the effects of Rc by subtracting it from z11.

z11′(f)=z11(f)−Rc

-   -   5) Compute stray capacitance C0 at highest frequency (fh).

C0=1/[2*pi*fh(hz)*imag(z11(fh))]

-   -   6) Compute Motional Inductance

Ls=(1/(4*pi))*(imag[z11(fs)]−imag[z11(fs−Δf)])/Δf

-   -   7) Compute Motional Resistance

Rs=real[z11′(fs)]

-   -   8) Compute Motional Capacitance

Cs=1/[(2*pi*fs)̂2*Ls]

-   -   9) Compute Q values

Q=1./(2*pi*Fs*C1*(Rs+Rc))

Next a Variable Standard Deviation Method can be employed to narrow theselection of resonators.

-   -   10) Find resonators that have Series Resonance defined    -   11) Find resonators having Q values >80    -   12) Find the subpopulation of resonators that have passed both        steps 10 AND 11.    -   13) Compute the mean and Standard Deviation of Resonance        frequencies measured from subpopulation of resonators from step        12    -   14) Compute the mean and Standard Deviation of resonators        calculated Contact Resistance from subpopulation of resonators        from step 12    -   15) Compute the mean and Standard Deviation of resonators        calculated Motional Resistance from subpopulation of resonators        from step 12    -   16) Compute the mean and Standard Deviation of resonators        calculated Motional Inductance from subpopulation of resonators        from step 12    -   17) Compute the mean and Standard Deviation of resonators        calculated Motional Capacitance from subpopulation of resonators        from step 12    -   18) Compute the mean and Standard Deviation of resonators        calculated Static Capacitance from subpopulation of resonators        from step 12

Once the mean and standard deviation are calculated as described aboundary condition on each parameter is applied according to thefollowing steps performed on the subpopulation of resonators from step12:

-   -   19) Compute the distribution of Contact Resistance    -   20) Compute the distribution of Motional Resistance    -   21) Compute the distribution of Motional Inductance    -   22) Compute the distribution of Motional Capacitance    -   23) Compute the distribution of Static Capacitance    -   24) Select varying SD value for each variable from steps 19-23    -   25) Find the subset of resonators that pass all user selected SD        boundary conditions in step 24    -   26) Compute the yield and visually inspect smith chart circles        for any outlying resonators    -   27) Tweak SD values for each variable in step 24 until all        undesirable resonators are excluded from the subpopulation.        One example implementation of the process of calculating Q        values is realized in the following code:

Ref_Coeff=10.{circumflex over ( )}(MA./20); RE=Ref_Coeff.*cos(PA.*pi/180);  IM=Ref_Coeff.*sin(PA.*pi/180); Admittance=(.02*(1−RE−j*IM)./(1+RE+j*IM));  Real_Adm=real (Admittance); Imag_Adm=imag (Admittance);   DEN=1−(2*RE) + (RE.*RE) + (IM.*IM);   REZ= (50*(1−(RE.*RE) − (IM.*IM)))./DEN;   IMZ = (50*2*IM)./DEN;   for L =1:1:length(RE(1,:))    QMAX = 0;    for I = 1:length(Freq)     if (I>=6)    A=((2*Freq(I)) / (Freq (5) − Freq (1)));     B=(REZ(I−5,L) * IMZ(I,L)) − (REZ(I,L)*IMZ (I − 5,L)):     C=(REZ(I,L) + REZ(I−5,L){circumflex over ( )} 2+(IMZ(I,L) + IMZ (I − 5,L)){circumflex over ( )}2;     Q(I,L) = A*B/C;     if (abs(Q(I,L)) >QMAX)      QMAX =abs(Q(I,L));      FMAX = Freq(I);     end    end   end  endFINALQ=[FINALQ Q];

The following process is an example for determining the quality factorof a resonator according to one embodiment:

Step 1: Compute Reflection Losses.

-   -   Ref_losses=10^((Mag/20));

Step 2: Compute the Reflections Coefficients

-   -   RE=Ref_losses.*cos(Phase.*pi/180);    -   IM=Ref_losses.*sin(Phase.*pi/180);

Step 3: Compute the Admittance from Reflection Coefficients

-   -   Admittance=(0.02*(1−RE−j*IM)./(1+RE+j*IM));    -   Real_Adm=real (Admittance);    -   Imag_Adm=imag (Admittance);

Step 4: Compute the Real and Imaginary IMP

-   -   DEN=1−(2*RE)+(RE.*RE)+(IM.*IM);    -   REZ=(50*(1−(RE.*RE)−(IM.*IM)))./DEN;    -   IMZ=(50*2*IM)./DEN;

Steps: Compute Q for each Frequency and each time point of measurementfor L=1:1:length (RE(1,:))

QMAX = 0;    for I=1:length(Freq)−1     if (I>=6)   A=(2*Freq(I))/(Freq(5) − Freq(1)));   B=(REZ(I−5,L)*IMZ(I,L))−(REZ(I,L)*IMZ(I−5,L));   C=(REZ(I,L)+REZ(I−5,L)){circumflex over( )}2+(IMZ(I,L)+IMZ(I−5L)){circumflex over ( )}2;    Q(I,L) = A*B/C;    if (abs(Q(I,L)) > QMAX)      QMAX=abs(Q(I,L));      FMAX=Freq(I);    end   end  end end

The method above can be further modified with the following equations toimprove computation speed:

$\begin{matrix}{Z = \frac{50*\left( {1 + {10^{({{Mag}/20})}*^{({iphase})}}} \right.}{\left( {1 - {10^{({{Mag}/20})}*^{({iphase})}}} \right)}} & 1 \\{{{Scaling}\mspace{14mu} {Factor}\text{:}\mspace{14mu} A} = {\left( {2*{Freq}} \right)/\left( {{{Freq}(5)} - {{Freq}(1)}} \right)}} & 2 \\{Q = {A*1.2*\frac{{{real}\; (Z)*{diff}\; \left( {{Imag}(Z)} \right)} - {{{{Imag}(Z)}.}*{{diff}\left( {{Real}\; (Z)} \right)}}}{{Abs}\; (Z)^{\hat{}2}}}} & 3\end{matrix}$

In the frequency shift detection scheme such as in the embodimentdescribed with reference to FIG. 5, the control circuit automaticallyadjusts the driving frequency of the sensor and reference resonators toalways maintain their resonant point, and the adjustment of the drivingfrequency is indicative of the change in resonance characteristic. Incontrast, the phase shift detection arrangement of the embodiments ofFIGS. 3 and 4 involves determining optimal driving frequency for thesensor and reference resonators.

A variety of ways to determine a resonator's optimal operating frequencyare contemplated. This process, referred to as tuning, or operatingpoint calibration, is carried out rapidly according to variousembodiments. This enables the use of phase shift detection schemes formeasuring samples having high concentrations of analyte.

In one approach, the determination of a resonator's operating frequencyis found by post-processing of gathered data, in which the group delayof phase for each signal frequency supplied to a resonator isdetermined, and the operating resonance frequency is defined as thefrequency of maximum group delay.

In another approach, the operating resonance frequency of the resonatorcan be efficiently determined in a calculation using measured actualoscillation characteristics represented by the real and imaginaryreflection coefficients. In this approach, a the frequency (f1) isdetermined at which Real(Z) has its maximum value and the frequency (f2)at which Imaginary(Z) has its maximum value. Using this approach, theoperating resonance frequency of the resonator is defined as the mean off1 and f2. This approach provides improved computational efficiency overpreviously-used techniques. In turn, the computational efficiency allowsfaster tuning, which can be extremely important in applications wherethe material being measured binds quickly to the resonant sensor.

In an example of how this calculation may be applied, a sensor iscoupled with the instrument and the instrument immediately tunes theresonator in air by sweeping through frequencies over a broad bandwidth.This initial tuning can be accomplished either by determining the groupdelay at each frequency and identifying the frequency at which themaximum group delay occurs, or by using the calculation at eachfrequency and finding the mean of f1 and f2 using the method disclosedabove. The in-air operating frequency is then used to define a narrowbandwidth, for example +/−5 MHz, or +/−1 MHz, of each resonator'soperating frequency, that will be used for a second tuning step that canbe carried out once the resonators are exposed to test sample. Thesecond tuning step is accomplished using the calculation methoddescribed for the initial tuning step, above, along with a finersampling interval than that used to sweep through the frequency windowused for the first tuning step while the sensor was in air.

In a related embodiment, an adaptive frequency sweep is performedinstead of a basic sweep using predetermined sampling intervals. Forinstance, at each operating frequency during the adaptive sweep, theoscillation characteristics (e.g., Real(Z) and Imaginary(Z)) aremeasured and compared to the corresponding values at previous orsubsequent frequencies. The result of this comparison determines if thedirection of the sweep is approaching, or moving away from the resonantpoint. This approach can produce a faster tuning of the resonator to itsresonant frequency since the full sweep can be avoided in some cases.

In various embodiments, the frequency sweeping, calculations,interpretation of measured parameters, and control of the tuning processis carried out by a controller interfaced with the sensing and measuringcircuitry and programmed to execute the tuning routine. The controllermay include a digital system having primarily hardware devices such asan application specific integrated circuit (ASIC) or field-programmablegate array (FPGA), for example, or may include a combination of hardwareand software, such as by a microprocessor system and a set ofinstructions to implement the controller's functionality. In otherembodiments, the controller can be implemented as a combination of thetwo, with certain functions facilitated by hardware alone, and otherfunctions facilitated by a combination of hardware and software. Avariety of suitable microprocessor systems may be utilized including,without limitation, one or more microcontrollers, one or more digitalsignal processors, and the like, along with appropriate interfacingcircuitry, data storage, power conditioning system, etc., as needed toimplement the controller's functionality.

Referring now to FIG. 9A, an efficient technique according to oneembodiment for accomplishing a tuning sweep in a short time isillustrated. A first tuning step (performed while the resonator is inair—i.e., not yet placed in the sample) performs a relatively wide sweeprange with a larger frequency step size setting for the VCO, orfrequency generator. Successive fine-tuning (performed after theresonator is placed in the sample) progressively reduce the frequencysweep range with progressively smaller frequency step size. The numberof steps that are employed is based on the assay characteristics andother variables such as microcontroller speed and bus communicationperformance. FIG. 9B is a diagram illustrating an example of such ascheme using an ADF4360 VCO as the frequency generator.

In a related embodiment, an even faster tuning is accomplished once thesensor is in a test sample with a modification of the second tuning stepusing a wider sampling interval that is intermediate between theintervals used in the first and second tuning steps in the exampledescribed above, to sweep through the +/−5 MHz window to identify, forexample, a 1 MHz window within which a much finer sampling interval isused to achieve a more precise tuning. Additional “nested” tuning stepsmay be used as needed and as hardware processing speeds allow to furtherimprove precision and speed of tuning.

This more efficient method can be important in certain applications inwhich the test sample binds very quickly to the sensor. When a sensorcomes into contact with a test sample the reaction of the capture ligandon the sensor surface with the target in the sample begins immediately.It is therefore desirable for the optimum resonator operatingfrequencies to be determined in the shortest possible time followingcontact with test sample. Using the narrow bandwidth determined whentuned in air and then using the technique disclosed above addresses thisneed in a way not previously described by enabling tuning to be achievedwithin one second or less of sample contact. In one embodiment, tuningwithin 10 milliseconds is achieved. In a further embodiment, tuningwithin 5 milliseconds is achieved. In yet another embodiment, withsufficient processing capability, sensor tuning is achieved in less thanone millisecond.

In one embodiment of the invention, the precise resonant frequency ofeach resonator is determined in air just prior to the introduction ofthe sensor into a test medium. By tuning the resonant frequency bysweeping through a range of approximately +/−3-5% of the resonatorfrequency prior to testing a first approximation of the resonantfrequency of each resonator can be achieved with the methods discussedabove used to analyze the response to the frequency sweep.

Additionally, in some embodiments an additional tuning process isperformed immediately after (and in response to) the introduction of thesample to the sensor according to the exemplary process discussed above.In one example embodiment of the invention, each resonator is tuned tobe driven at its ideal resonant frequency well within the first secondof being introduced into the test medium. By tuning the resonantfrequency by sweeping through a narrow range, for example, +/−5 MHz ofthe first approximation resonant frequency, a more exact resonantfrequency can be used for testing. Other ranges may be appropriatedepending on the base frequency of the resonator.

This in situ tuning has the advantage of further refining the drivingresonant frequency of each resonator to take into account the transitionfrom air to the test medium, such as a liquid solution. In the casewhere the target substance was known to instantly bind to a coating onthe sensing resonator it may not be possible to perform an in situtuning, but presently known coatings and target substances do providemany examples where the binding reaction can take longer than the timeneeded to perform the in situ tuning.

Examples of the operation of phase-shift and frequency-shift detectorembodiments of instrument 12 will now be described with reference toFIGS. 10A-11B

FIGS. 10A and 10B are graphs depicting evolution of biosensor signalsover time during system operation for a frequency shift detectionbiosensor and a phase shift detection biosensor, respectively, accordingto embodiments of the invention. The biosensor signal may be phase,frequency, or some other metric indicative of the resonant behavior ofthe biosensor. The biosensor in these examples is intended formeasurement of an analyte in liquid samples.

Referring first to FIG. 10A, initially, the biosensor is exposed to air(similarly, the biosensor may initially be exposed to another medium).At time t1 operation of the system is initiated either by application ofpower, automatic detection of biosensor connection, external activationby a user, or other such mechanism. After initiation at time t1 thebiosensor output indicated at 90 is monitored by the electronics of thesystem while still in air or original medium (which is not the sample).

As detailed above, the monitoring apparatus according to variousembodiments generally comprises a microcontroller-based system or thelike that collects the biosensor signal at regular intervals. Themonitoring apparatus may or may not record the collected biosensorsignal. The monitoring apparatus continues to monitor the baselinebiosensor output until an abrupt step change 91 is measured. The abruptstep change occurs due to introduction of a liquid sample into contactwith the biosensor surface, causing the resonant properties of thebiosensor to shift in response to the change in medium viscosity. For agiven biosensor of a known construction, the approximate magnitude ofthe abrupt change will be known for a transition from air to liquidmedium, thus providing a threshold, or target range of values, theachievement of which is deemed to be a sample introduction event. Usingthresholding or a target range of values provides an advantage ofignoring small changes or noise in order to avoid false indications.

Thus, at time t2, liquid sample with unknown analyte concentration isplaced in physical contact with the biosensor. In one embodiment, theabrupt biosensor signal step change 91 is used as a time referencerepresenting the onset of liquid sample exposure on the biosensorsurface. The abrupt signal change 91 will typically occur in less than 1second, and often occurs in less than 0.1 seconds of sampleintroduction. Once the abrupt step change 91 is detected, thebiosensor's output is monitored to measure the binding kinetics ofanalyte on the surface of the biosensor, as indicated at 92. In thisembodiment tuning of the resonator is automatically handled by a controlcircuit. Therefore, measurement of the binding kinetics can begin assoon as the output is stabilized in response to introduction of thesample. This stabilization can occur in a matter of milliseconds, and insome embodiments in less than one millisecond.

The binding of analyte on the surface can be used to determineconcentration of the analyte in the sample by any of a multitude ofmethods. One such method according to one embodiment involves measuringthe rate of change of the biosensor output at stage 92. When measuringanalyte concentration by rate of change, it is preferable according toone embodiment to measure the initial rate of change over a short timeperiod very soon after the liquid sample is initially introduced to thebiosensor surface. This early-stage measurement is commonly referred toas the initial on rate. As discussed above, especially for highconcentrations of analyte and/or high association rates between analyteand capture ligand on the biosensor surface, an electronic timeregistration of initial liquid sample contact with biosensor surface(from abrupt biosensor output transient 91 at time t2) provides improvedmeasurement accuracy and ability to measure faster binding rates.

The slope of biosensor output at 92 is proportional to the rate ofanalyte binding events on the biosensor surface. Over a range of analyteconcentrations, the rate of analyte binding events on the biosensorsurface is related to the concentration of analyte in the liquid sample.This relationship may be proportional or nonlinear, althoughproportionality may be preferred for the sake of accuracy and simplicityin practical embodiments. In this manner, the system uses accurateregistration of time t2 to derive an accurate measure of analyteconcentration in liquid sample that is accurate over a broad range ofanalyte concentrations.

A second method of determining analyte concentration according to arelated embodiment is to measure the total amount of analyte bound overa fixed time interval t3-t2 by the total change of biosensor output(i.e., the level at time t3 minus the initial level at time t2). Whenmeasuring analyte concentration by total change over fixed time intervalit is preferable to have an accurate measure of the initial start timefor interaction between analyte and biosensor. As the fixed test timeinterval becomes smaller (for intentionally reduced test times such asin an emergency situations or for high analyte concentrations) accuratemeasurement of analyte and biosensor interaction time becomesincreasingly important for making accurate analyte concentrationmeasurements. Referencing the initial time of the time interval to thedetected step change, the system of this embodiment uses accurateregistration of time t2 to derive an accurate measure of analyteconcentration in liquid sample that is accurate over a broad range ofanalyte concentrations.

FIG. 10B is a graph depicting similar operation using a phase detectionarrangement such as the embodiments described above with reference toFIG. 3 and FIG. 4. At time t4, operation of the system is initiated(e.g., by application of power, automatic detection of biosensorconnection, external activation by a user, or other means). Afterinitiation at time t4 the system begins performing a tuning of thebiosensor. This can be done for purposes of verifying proper function oroptimizing operation at a preferred condition such as determining aresonant frequency. The tuning begins at time t5 and completes at timet6. The biosensor output at 94 can be logged as part of the test record,and may be useful in ascertaining the condition of the resonator orinstrument. In a related embodiment, the interval between t4 and t5 maybe eliminated with initial activation comprising the tuning operation.

Based on tuning of the biosensor, the desired operating condition is setfor optimal operation at time t6 and the biosensor output at 95 ismonitored by the electronics of the system while still in the originalmedium (e.g., air). The monitoring apparatus continues to monitor thebiosensor output until an abrupt step change 96 is measured. At time t7,a liquid sample with unknown analyte concentration is placed in physicalcontact with the biosensor. Once the abrupt transient 96 is detected,the system performs a re-tuning for operation in the liquid sample. There-tuning is performed between times t7 and t8. At time t8 the operatingcondition is set for optimal operation (e.g., at the point of resonance)and the biosensor output at 97 is monitored to measure the bindingkinetics of analyte on the surface of the biosensor.

Particularly for high concentrations of analyte and/or high associationrates between analyte and capture ligand on biosensor surface (i.e.,avidity), an electronic time registration of initial liquid samplecontact with biosensor surface (from abrupt biosensor output transient96 at time t7) provides improved measurement accuracy and ability tomeasure faster binding rates. Furthermore, it is preferable for there-tuning to be performed in a short period of time such that initial onrate is not missed during the process of re-tuning. In one embodiment,the re-tuning is performed in less than 1 second, and more preferably,the re-tuning is performed in less than 0.1 second. In general, theslope of biosensor output 97 is proportional to the rate of analytebinding events on the biosensor surface. Over a range of analyteconcentrations, the rate of analyte binding events on the biosensorsurface is related to the concentration of analyte in the liquid sample(preferably, in proportion). In this manner, the system uses accurateregistration of time t7 and rapid re-tuning to derive an accuratemeasure of analyte concentration in liquid sample that is accurate overa broad range of analyte concentrations.

The second method of determining analyte concentration by measuring thetotal amount of analyte bound over a fixed time interval t9-t8 is alsoavailable in the phase change detection instrument. When measuringanalyte concentration by total change over the fixed time interval, itis preferable to have an accurate measure of the initial start time forinteraction between analyte and biosensor. It is further preferable tohave minimal analyte-biosensor exposure time consumed by the re-tuning.As the fixed test time interval becomes smaller (for intentionallyreduced test times such as in an emergency situation or for high analyteconcentrations) accurate measurement of analyte and biosensorinteraction time becomes increasingly important for making accurateanalyte concentration measurements. In this manner, the system accordingto this embodiment uses accurate registration of time t8 and rapidre-tuning to derive an accurate measure of analyte concentration inliquid sample that is accurate over a broad range of analyteconcentrations.

FIGS. 11A and 11B are a flow diagrams illustrating an exemplaryoperation, respectively, of systems that control and monitor thefrequency shift detection biosensor, and the phase shift detectionbiosensor, the outputs of which are represented in FIGS. 10A and 10Babove according to embodiments of the invention. In various embodiments,the block functions in FIGS. 11A and 11B are carried out under programcontrol, e.g., as microcontroller firmware executed on a microcontrolleror digital signal processor, with characteristic execution speed relatedto the circuit's architecture and clock frequency. Data acquisitionoperations such as block 104 that measures and records biosensor outputmay also involve analog-to-digital conversions (ADC) requiring multipleclock cycles or delays. In general, it is contemplated that each of theblocks can be executed in much less than 1 second, and preferably lessthan 1 millisecond. Fast electronic operation through each functionalblock provides for high accuracy measurements of analyte concentrationin liquid samples.

Referring to FIG. 11A, operation of the system is initiated at 102,either by application of power, automatic detection of biosensorconnection, external activation by a user, or other such mechanism. At104, the system monitors and records output from the biosensor,typically measuring resonant frequency or phase angle offset relative tothe driving signal. After each measurement, the system compares the mostrecent measurement to a previous measurement to determine if an abruptbiosensor signal change has occurred at decision 106. A choice function(typically including the difference between current and a previousmeasurement compared against a threshold or target range of minimum andmaximum values corresponding to the anticipated step change range ofvalues) is executed to decide if liquid sample has come into contactwith the biosensor surface.

If an abrupt biosensor signal change has not occurred, execution loopsback to monitoring and recording of additional biosensor output valuesat block 104. The 106-104 loop performs a “wait” operation in which thesystem monitors the biosensors output signal and waits for an abruptchange in the biosensor signal indicative of transition from air toliquid medium at the biosensor interface. The loop time constant, orrepetition rate, is generally less than 1 second, and preferably lessthan 1 millisecond. Smaller loop time constants provide for moreaccurate measurement of the initial time of liquid introduction onbiosensor surface.

If an abrupt biosensor signal change meeting the threshold or othercriteria has occurred, execution control proceeds to 108 to monitor thebinding of analyte on the biosensor surface. Here, the system executescontinuous monitoring of the biosensor signal output in the loop110-108. After each measurement in block 108, the total time of liquidexposure is evaluated in decision 110 to determine if the test iscomplete. For example, in the operation depicted in FIG. 10A, the testis completed at time t3. This 110-108 loop has a characteristicperiodicity in time, or repetition rate that is typically less than 1second, and preferably less than 1 millisecond. Once the desired testtime is complete, the process continues to block 112, where analyteconcentration is calculated based on the recorded data, either frominitial rate of change or as final endpoint value (i.e., 93 in FIG.10A). Calculation of analyte concentration may include recording ortransmission of data, followed by transfer to execution control totermination or completion of the program at 114.

Referring now to FIG. 11B, a similar process is depicted for a phaseshift detection biosensor. The numbered blocks 102-114 represent thecorrespondingly numbered blocks described above with reference to FIG.11A. In this process, however, the additional sub-processes of tuningare performed at 116 and 118. Tuning is performed in air at 116 prior toinitiating the measure and record operation at 104. The placement ofthis first tuning operation at this stage can maximize the sensor'ssensitivity so that comparisons against the threshold at 106 in thedetermination of the step change are trustworthy. In one embodiment, thefirst tuning operation is performed immediately in response toinitiation of operation.

The second tuning operation takes place in response to the detection ofexposure of the resonator to the liquid sample at 106. In a preferredembodiment, this second tuning operation at 118 is performed as soon aspossible upon the detection of the step change. The second tuningoperation can be performed in well under one second, and preferablywithin a millisecond of the detection of the step change. The durationof the tuning procedure itself should also be performed in as short atime as practicable according to one embodiment. Thus, for instance, thetuning at 118 is performed within one second and, preferably within 0.1second. Even more preferably, the tuning at 118 is accomplished within50 milliseconds. For instance, in the embodiment described above withreference to FIG. 9B, tuning is accomplished in 37.5 milliseconds. It iscontemplated that advances in processing capacity and in analogmicroelectronics will permit faster performance of the tuningoperations.

In a related embodiment, the measure and record sensor output operationat 108 is initiated immediately, i.e., as fast as possible, in responseto completion of the tuning operation at 118. In a practical embodimentthis should be done within one second, and preferably within onemillisecond. The first and the second tuning operations 116 and 118, invarious embodiments, can be performed as detailed above with referenceto FIGS. 9A and 9B.

In a related embodiment, where a sensing and reference resonator areutilized, the tuning operations 116 and 118 are performed on each of theresonators. In one such system, the resonators are tuned at about thesame time, rather than sequentially one after the other.

In another sensing-and-reference resonator embodiment, the individualresonator output of either the sensing resonator alone, the referenceresonator alone, or of both resonators in common mode (i.e., notdifferential mode), is monitored to detect the introduction of thesample. Measurement of binding kinetics, on the other hand, is based onthe differential mode between the sensor and reference devices, asdiscussed above.

More generally, the step change detected at 106 more generallyrepresents a change from one fluid viscosity characteristic to another.For instance the change can be from liquid to a gas, or from a liquid ofa first density to a liquid of a second, different, density. In arelated embodiment, the instrument is configured with an appropriatethreshold or step change value target range corresponding to theexpected change in viscosity from initiation of operation at 104 andintroduction of the sample. This information can be stored, forinstance, along with the calibration constants or other parametersspecific to the biosensor assembly or test protocol in a data storagedevice such as the one described above, or in a remote but accessiblelocation such as a personal computer workstation or server.

The embodiments above are intended to be illustrative and not limiting.Additional embodiments are within the claims. In addition, althoughaspects of the present invention have been described with reference toparticular embodiments, those skilled in the art will recognize thatchanges can be made in form and detail without departing from the scopeof the invention, as defined by the claims.

Persons of ordinary skill in the relevant arts will recognize that theinvention may comprise fewer features than illustrated in any individualembodiment described above. The embodiments described herein are notmeant to be an exhaustive presentation of the ways in which the variousfeatures of the invention may be combined. Accordingly, the embodimentsare not mutually exclusive combinations of features; rather, theinvention may comprise a combination of different individual featuresselected from different individual embodiments, as will be understood bypersons of ordinary skill in the art.

Any incorporation by reference of documents above is limited such thatno subject matter is incorporated that is contrary to the explicitdisclosure herein. Any incorporation by reference of documents above isfurther limited such that no claims that are included in the documentsare incorporated by reference into the claims of the presentApplication. The claims of any of the documents are, however,incorporated as part of the disclosure herein, unless specificallyexcluded. Any incorporation by reference of documents above is yetfurther limited such that any definitions provided in the documents arenot incorporated by reference herein unless expressly included herein.

For purposes of interpreting the claims for the present invention, it isexpressly intended that the provisions of Section 112, sixth paragraphof 35 U.S.C. are not to be invoked unless the specific terms “means for”or “step for” are recited in a claim.

What is claimed is:
 1. A method for measuring binding kinetics of aninteraction of an analyte material present in a fluid sample with one ormore resonating devices, at least one of which is a sensing resonatorhaving binding sites for the analyte material, the method comprising:prior to exposing the one or more resonating devices to the fluidsample, initiating operation of the one or more resonating devices thatproduces one or more resonator output signals representing a resonancecharacteristic of each of the one or more of the resonating sensors;automatically detecting introduction of a fluid sample to the one ormore resonating devices based on detection of a characteristic change inthe one or more resonator output signals; in response to the detectingof the introduction of the fluid sample, initiating automatedmeasurement of the binding kinetics of the analyte material to the atleast one resonating sensor having the binding sites.
 2. The method ofclaim 1, wherein the automatically detecting of the introduction of thefluid sample to the one or more resonating devices includes detecting achange in viscosity of a fluid in physical contact with a surface of theone or more resonating devices.
 3. The method of claim 1, whereininitiating the automated measurement of the binding kinetics includesmonitoring of the one or more resonator output signals from a timereference based on the time of occurrence of the characteristic changein the one or more resonator output signals.
 4. The method of claim 1,wherein the detection of the characteristic change in the one or moreresonator output signals includes detection of a step change in aresonant characteristic of the one or more resonating devices selectedfrom the group consisting of: a frequency, a phase angle, or anycombination thereof.
 5. The method of claim 1, wherein initiatingoperation of the one or more resonating devices includes operating theone or more resonating devices in ambient air, and wherein the fluidsample is a liquid.
 6. The method of claim 1, wherein the fluid sampleis an unrefined biological sample.
 7. The method of claim 1, whereininitiating automated measurement of the binding kinetics includesinitiating measurement of a rate of change of the one or more resonatoroutput signals within one second of the detecting of the introduction ofthe fluid sample.
 8. The method of claim 7, wherein initiating automatedmeasurement of the binding kinetics includes measuring a rate of changeof the one or more resonator output signals within 0.1 second of thedetecting of the introduction of the fluid sample.
 9. The method ofclaim 7, wherein initiating automated measurement of the bindingkinetics includes measuring a rate of change of the one or moreresonator output signals within 0.05 second of the detecting of theintroduction of the fluid sample.
 10. The method of claim 1, whereininitiating automated measurement of the binding kinetics includesmeasuring a total amount of change of the one or more resonator outputsignals time-referenced from the detection of the characteristic changein the one or more resonator output signals.
 11. The method of claim 1,wherein the one or more resonating devices includes at least onereference resonator that lacks any binding sites for the analytematerial, and wherein initiating measurement of the binding kineticsincludes measuring a difference between at least one sensor outputsignal of the at least one sensing resonator and at least one referenceoutput signal of the at least one reference resonator.
 12. The method ofclaim 11, further comprising operating each of the at least one sensingresonator and the at least one reference resonator at theircorresponding resonant frequencies, which are potentially different fromone another.
 13. The method of claim 1, further comprising: based on thebinding kinetics, determining a measure of concentration of the analytein the fluid sample.
 14. The method of claim 1, wherein automaticallydetecting introduction of a fluid sample to the one or more resonatingdevices includes comparing a degree of the characteristic change in theone or more resonator output signals against a threshold value or targetrange of values such that false detections are suppressed.